Plant regulatory elements from a metallothionein-like gene and uses thereof

ABSTRACT

The present invention provides novel regulatory elements for use in plants. The present invention also provides DNA constructs containing these novel regulatory elements; transgenic cells, plants, and seeds containing these novel regulatory elements; and methods for preparing and using the same.

This application claims the benefit of U.S. provisional application No.61/042,957 filed Apr. 7, 2008, herein incorporated by reference in itsentirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“MONS222WO_Sequence_Listing”, which is 30.2 Kbytes (as measured inMicrosoft Windows®) and was created on Mar. 30, 2009, is filed herewithby electronic submission and is incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to the field of plant molecular biology and plantgenetic engineering and DNA molecules useful for modulating geneexpression in plants.

BACKGROUND

Regulatory elements are genetic elements that regulate gene activity bymodulating the transcription of an operably linked transcribablepolynucleotide molecule. Such elements include promoters, leaders,introns, and 3′ untranslated regions and are useful in the field ofplant molecular biology and plant genetic engineering.

SUMMARY OF THE INVENTION

The present invention provides novel regulatory elements from Foxtailmillet (Setaria italica (L.) Beauv) for use in plants. The presentinvention also provides DNA constructs comprising the regulatoryelements. The present invention also provides transgenic plant cells,plants, and seeds comprising the regulatory elements operably linked toa transcribable polynucleotide molecule. The present invention alsoprovides methods of making and using the regulatory elements, the DNAconstructs comprising the regulatory elements, and the transgenic plantcells, plants, and seeds comprising the regulatory elements operablylinked to a transcribable polynucleotide molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the three promoter variants designed from theregulatory elements from the Lipid Transfer Protein gene. SEQ ID NO: 15is P-SETit.Rcc3-1:1:1 and is 2062 nucleotide base pairs in length; SEQID NO: 20 is P-SETit.Rcc3-1:1:11 and is 915 nucleotide base pairs inlength; and SEQ ID NO: 18 is P-SETit.Rcc3-1:1:10 and is 1563 nucleotidebase pairs in length.

FIG. 2 illustrates the two promoter variants designed from theregulatory elements from the Metallothionein-like protein gene. SEQ IDNO: 5 is P-SETit.Mtha-1:1:1 and is 483 base pairs long; SEQ ID NO: 8 isP-SETit.Mthb-1:1:2 and is 1516 base pairs in length.

FIGS. 3A and 3B collectively illustrate a sequence alignment producedusing CLUSTAL W (1.82) multiple sequence alignment of the two allelicvariants of the promoters from the Dehydration Related Protein gene. Inthe consensus below the aligned sequences, matches are marked with “*”,mismatches are marked with “.”, and deletions/insertions are marked with“-”. The two allelic variants had identical leader sequences, but thepromoter sequences were sequence variants when aligned. SEQ ID NO: 10 isP-SETit.DRPa-1:1:1 and SEQ ID NO: 13 is P-SETit.DRPb-1:1:1.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

DNA Molecules

As used herein, the term “DNA” or “DNA molecule” refers to adouble-stranded DNA molecule of genomic or synthetic origin, i.e., apolymer of deoxyribonucleotide bases or a polynucleotide molecule, readfrom the 5′ (upstream) end to the 3′ (downstream) end. As used herein,the term “DNA sequence” refers to the nucleotide sequence of a DNAmolecule. The nomenclature used herein is that required by Title 37 ofthe United States Code of Federal Regulations §1.822 and set forth inthe tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

As used herein, the term “isolated DNA molecule” refers to a DNAmolecule at least partially separated from other molecules normallyassociated with it in its native or natural state. In one embodiment,the term “isolated” refers to a DNA molecule that is at least partiallyseparated from the nucleic acids which normally flank the DNA moleculein its native or natural state. Thus, DNA molecules fused to regulatoryor coding sequences with which they are not normally associated, forexample as the result of recombinant techniques, are considered isolatedherein. Such molecules are considered isolated even when integrated intothe chromosome of a host cell or present in a nucleic acid solution withother DNA molecules.

Any number of methods well known to those skilled in the art can be usedto isolate and manipulate a DNA molecule, or fragment thereof, disclosedin the present invention. For example, PCR (polymerase chain reaction)technology can be used to amplify a particular starting DNA moleculeand/or to produce variants of the original molecule. DNA molecules, orfragment thereof, can also be obtained by other techniques such as bydirectly synthesizing the fragment by chemical means, as is commonlypracticed by using an automated oligonucleotide synthesizer.

As used herein, the term “sequence identity” refers to the extent towhich two optimally aligned polynucleotide sequences are identical. Anoptimal sequence alignment is created by manually aligning twosequences, e.g. a reference sequence and another sequence, to maximizethe number of nucleotide matches in the sequence alignment withappropriate internal nucleotide insertions, deletions, or gaps. As usedherein, the term “reference sequence” refers to a sequence provided asSEQ ID NO: 1-20.

As used herein, the term “percent sequence identity” or “percentidentity” or “% identity” is the identity fraction times 100. The“identity fraction” for a sequence optimally aligned with a referencesequence is the number of nucleotide matches in the optimal alignment,divided by the total number of nucleotides in the reference sequence,e.g. the total number of nucleotides in the full length of the entirereference sequence. Thus, one embodiment of the invention is a DNAmolecule comprising a sequence that when optimally aligned to areference sequence, provided herein as SEQ ID NO: 1-20, has about 85percent identity or higher, about 90 percent identity or higher, about95 percent identity or higher, or at least 96 percent identity, 97percent identity, 98 percent identity, or 99 percent identity to thereference sequence and has gene regulatory activity.

Regulatory Elements

A regulatory element is a DNA molecule having gene regulatory activity,i.e. one that has the ability to affect the transcription and/ortranslation of an operably linked transcribable polynucleotide molecule.The term “gene regulatory activity” thus refers to the ability to affectthe expression pattern of an operably linked transcribablepolynucleotide molecule by affecting the transcription and/ortranslation of that operably linked transcribable polynucleotidemolecule. Gene regulatory activity may be positive and/or negative andthe effect may be characterized by its temporal, spatial, developmental,tissue, environmental, physiological, pathological, cell cycle, and/orchemically responsive qualities as well as by quantitative orqualitative indications.

Regulatory elements such as promoters, leaders, introns, andtranscription termination regions are DNA molecules that have generegulatory activity and play an integral part in the overall expressionof genes in living cells. The term “regulatory element” refers to a DNAmolecule having gene regulatory activity, i.e. one that has the abilityto affect the transcription and/or translation of an operably linkedtranscribable polynucleotide molecule. Isolated regulatory elements,such as promoters and leaders, that function in plants are thereforeuseful for modifying plant phenotypes through the methods of geneticengineering.

Regulatory elements may be characterized by their expression pattern,i.e. as constitutive and/or by their temporal, spatial, developmental,tissue, environmental, physiological, pathological, cell cycle, and/orchemically responsive expression pattern, and any combination thereof,as well as by quantitative or qualitative indications. A promoter isuseful as a regulatory element for modulating the expression of anoperably linked transcribable polynucleotide molecule.

As used herein, a “gene expression pattern” is any pattern oftranscription of an operably linked DNA molecule into a transcribed RNAmolecule. Expression may be characterized by its temporal, spatial,developmental, tissue, environmental, physiological, pathological, cellcycle, and/or chemically responsive qualities as well as by quantitativeor qualitative indications. The transcribed RNA molecule may betranslated to produce a protein molecule or may provide an antisense orother regulatory RNA molecule, such as a dsRNA, a tRNA, an rRNA, amiRNA, and the like.

As used herein, the term “protein expression is any pattern oftranslation of a transcribed RNA molecule into a protein molecule.Protein expression may be characterized by its temporal, spatial,developmental, or morphological qualities as well as by quantitative orqualitative indications.

As used herein, the term “promoter” refers generally to a DNA moleculethat is involved in recognition and binding of RNA polymerase II andother proteins (trans-acting transcription factors) to initiatetranscription. A promoter may be initially isolated from the 5′untranslated region (5′ UTR) of a genomic copy of a gene. Alternately,promoters may be synthetically produced or manipulated DNA molecules.Promoters may also be chimeric, that is a promoter produced through thefusion of two or more heterologous DNA molecules. Promoters useful inpracticing the present invention include SEQ ID NO: 2, 5, 8, 10, 13, and20 or fragments or variants thereof.

In one embodiment, fragments are provided of a promoter sequencedisclosed herein. Promoter fragments may exhibit promoter activity, andmay be useful alone or in combination with other promoters and promoterfragments, such as in constructing chimeric promoters. In specificembodiments, fragments of a promoter are provided comprising at leastabout 50, 95, 150, 250, 500, or about 750 contiguous nucleotides of apolynucleotide molecule having promoter activity disclosed herein. Suchfragments may exhibit at least about 85 percent, about 90 percent, about95 percent, about 98 percent, or about 99 percent, or greater, identitywith a reference sequence when optimally aligned to the referencesequence.

A promoter or promoter fragment may also be analyzed for the presence ofknown promoter elements, i.e. DNA sequence characteristics, such as aTATA-box and other known transcription factor binding site motifs.Identification of such known promoter elements may be used by one ofskill in the art to design variants of the promoter having a similarexpression pattern to the original promoter.

As used herein, the term “enhancer” or “enhancer element” refers to acis-acting transcriptional regulatory element, a.k.a. cis-element, whichconfers an aspect of the overall expression pattern, but is usuallyinsufficient alone to drive transcription, of an operably linkedpolynucleotide sequence. Unlike promoters, enhancer elements do notusually include a transcription start site (TSS) or TATA box. A promotermay naturally comprise one or more enhancer elements that affect thetranscription of an operably linked polynucleotide sequence. An isolatedenhancer element may also be fused to a promoter to produce a chimericpromoter.cis-element, which confers an aspect of the overall modulationof gene expression. A promoter or promoter fragment may comprise one ormore enhancer elements that effect the transcription of operably linkedgenes. Many promoter enhancer elements are believed to bind DNA-bindingproteins and/or affect DNA topology, producing local conformations thatselectively allow or restrict access of RNA polymerase to the DNAtemplate or that facilitate selective opening of the double helix at thesite of transcriptional initiation. An enhancer element may function tobind transcription factors that regulate transcription. Some enhancerelements bind more than one transcription factor, and transcriptionfactors may interact with different affinities with more than oneenhancer domain. Enhancer elements can be identified by a number oftechniques, including deletion analysis, i.e., deleting one or morenucleotides from the 5′ end or internal to a promoter; DNA bindingprotein analysis using DNase I footprinting, methylation interference,electrophoresis mobility-shift assays, in vivo genomic footprinting byligation-mediated PCR, and other conventional assays; or by DNA sequencesimilarity analysis using known cis-element motifs or enhancer elementsas a target sequence or target motif with conventional DNA sequencecomparison methods, such as BLAST. The fine structure of an enhancerdomain can be further studied by mutagenesis (or substitution) of one ormore nucleotides or by other conventional methods. Enhancer elements canbe obtained by chemical synthesis or by isolation from regulatoryelements that include such elements, and they can be synthesized withadditional flanking nucleotides that contain useful restriction enzymesites to facilitate subsequence manipulation. Thus, the design,construction, and use of enhancer elements according to the methodsdisclosed herein for modulating the expression of operably linkedtranscribable polynucleotide molecules are encompassed by the presentinvention.

As used herein, the term “leader” refers to a DNA molecule isolated fromthe untranslated 5′ region (5′ UTR) of a genomic copy of a gene anddefined generally as a nucleotide segment between the transcriptionstart site (TSS) and the protein coding sequence start site.Alternately, leaders may be synthetically produced or manipulated DNAelements. A leader can be used as a 5′ regulatory element for modulatingexpression of an operably linked transcribable polynucleotide molecule.Leader molecules may be used with a heterologous promoter or with theirnative promoter. Promoter molecules of the present invention may thus beoperably linked to their native leader or may be operably linked to aheterologous leader. Leaders useful in practicing the present inventioninclude SEQ ID NO: 3, 6, 11, and 16 or fragments or variants thereof.

As used herein, the term “chimeric” refers to a single DNA moleculeproduced by fusing a first DNA molecule to a second DNA molecule, whereneither first nor second DNA molecule would normally be found in thatconfiguration, i.e. fused to the other. The chimeric DNA molecule isthus a new DNA molecule not otherwise normally found in nature. As usedherein, the term “chimeric promoter” refers to a promoter producedthrough such manipulation of DNA molecules. A chimeric promoter maycombine two or more DNA fragments; an example would be the fusion of apromoter to an enhancer element. Thus, the design, construction, and useof chimeric promoters according to the methods disclosed herein formodulating the expression of operably linked transcribablepolynucleotide molecules are encompassed by the present invention.

As used herein, the term “variant” refers to a second DNA molecule thatis in composition similar, but not identical to, a first DNA moleculeand yet the second DNA molecule still maintains the generalfunctionality, i.e. same or similar expression pattern, of the first DNAmolecule. A variant may be a shorter or truncated version of the firstDNA molecule and/or an altered version of the sequence of the first DNAmolecule, such as one with different restriction enzyme sites and/orinternal deletions, substitutions, and/or insertions. In the presentinvention, a polynucleotide sequence provided as SEQ ID NO: 1-20 may beused to create variants that are in composition similar, but notidentical to, the polynucleotide sequence of the original regulatoryelement, while still maintaining the general functionality, i.e. same orsimilar expression pattern, of the original regulatory element.Production of such variants of the present invention is well within theordinary skill of the art in light of the disclosure and is encompassedwithin the scope of the present invention.

Constructs

As used herein, the term “construct” means any recombinantpolynucleotide molecule such as a plasmid, cosmid, virus, autonomouslyreplicating polynucleotide molecule, phage, or linear or circularsingle-stranded or double-stranded DNA or RNA polynucleotide molecule,derived from any source, capable of genomic integration or autonomousreplication, comprising a polynucleotide molecule where one or morepolynucleotide molecule has been linked in a functionally operativemanner, i.e., operably linked. As used herein, the term “vector” meansany recombinant polynucleotide construct that may be used for thepurpose of transformation, i.e., the introduction of heterologous DNAinto a host cell.

As used herein, the term “operably linked” refers to a first moleculejoined to a second molecule, wherein the molecules are so arranged thatthe first molecule affects the function of the second molecule. The twomolecules may or may not be part of a single contiguous molecule and mayor may not be adjacent. For example, a promoter is operably linked to atranscribable polynucleotide molecule if the promoter modulatestranscription of the transcribable polynucleotide molecule of interestin a cell.

The constructs of the present invention are generally double Ti plasmidborder DNA constructs that have the right border (RB or AGRtu.RB) andleft border (LB or AGRtu.LB) regions of the Ti plasmid isolated fromAgrobacterium tumefaciens comprising a T-DNA, that along with transfermolecules provided by the Agrobacterium tumefaciens cells, permit theintegration of the T-DNA into the genome of a plant cell (see, forexample, U.S. Pat. No. 6,603,061). The constructs may also contain theplasmid backbone DNA segments that provide replication function andantibiotic selection in bacterial cells, for example, an Escherichiacoli origin of replication such as ori322, a broad host range origin ofreplication such as oriV or oriRi, and a coding region for a selectablemarker such as Spec/Strp that encodes for Tn7 aminoglycosideadenyltransferase (aadA) conferring resistance to spectinomycin orstreptomycin, or a gentamicin (Gm, Gent) selectable marker gene. Forplant transformation, the host bacterial strain is often Agrobacteriumtumefaciens ABI, C58, or LBA4404; however, other strains known to thoseskilled in the art of plant transformation can function in the presentinvention.

Methods are known in the art for assembling and introducing constructsinto a cell in such a manner that the transcribable polynucleotidemolecule is transcribed into a functional mRNA molecule that istranslated and expressed as a protein product. For the practice of thepresent invention, conventional compositions and methods for preparingand using constructs and host cells are well known to one skilled in theart, see, for example, Molecular Cloning: A Laboratory Manual, 3^(rd)edition Volumes 1, 2, and 3 (2000) J. F. Sambrook, D. W. Russell, and N.Irwin, Cold Spring Harbor Laboratory Press. Methods for makingrecombinant vectors particularly suited to plant transformation include,without limitation, those described in U.S. Pat. Nos. 4,971,908;4,940,835; 4,769,061; and 4,757,011 in their entirety. These types ofvectors have also been reviewed in the scientific literature (see, forexample, Rodriguez, et al., Vectors: A Survey of Molecular CloningVectors and Their Uses, Butterworths, Boston, (1988) and Glick, et al.,Methods in Plant Molecular Biology and Biotechnology, CRC Press, BocaRaton, Fla. (1993)). Typical vectors useful for expression of nucleicacids in higher plants are well known in the art and include vectorsderived from the tumor-inducing (Ti) plasmid of Agrobacteriumtumefaciens (Rogers, et al., Methods in Enzymology, 153: 253-277(1987)). Other recombinant vectors useful for plant transformation,including the pCaMVCN transfer control vector, have also been describedin the scientific literature (see, for example, Fromm, et al., Proc.Natl. Acad. Sci. USA, 82: 5824-5828 (1985)).

Various regulatory elements may be included in a construct. Any suchregulatory elements may be provided in combination with other regulatoryelements. Such combinations can be designed or modified to producedesirable regulatory features. Constructs of the present invention wouldtypically comprise at least one regulatory element operably linked to atranscribable polynucleotide molecule operably linked to a 3′transcription termination molecule.

Constructs of the present invention may include any promoter or leaderknown in the art. For example, a promoter of the present invention maybe operably linked to a heterologous non-translated 5′ leader such asone derived from a heat shock protein gene (see, for example, U.S. Pat.Nos. 5,659,122 and 5,362,865). Alternatively, a leader of the presentinvention may be operably linked to a heterologous promoter such as theCauliflower Mosaic Virus 35S transcript promoter (see, U.S. Pat. No.5,352,605).

As used herein, the term “intron” refers to a DNA molecule that may beisolated or identified from the genomic copy of a gene and may bedefined generally as a region spliced out during mRNA processing priorto translation. Alternately, an intron may be a synthetically producedor manipulated DNA element. An intron may contain elements enhancerelements that effect the transcription of operably linked genes. Anintron may be used as a regulatory element for modulating expression ofan operably linked transcribable polynucleotide molecule. A DNAconstruct may comprise an intron, and the intron may or may not beheterologous with respect to the transcribable polynucleotide moleculesequence. Examples of introns in the art include the rice actin intron(U.S. Pat. No. 5,641,876) and the corn HSP70 intron (U.S. Pat. No.5,859,347).

As used herein, the term “3′ transcription termination molecule” or “3′UTR” refers to a DNA molecule that is used during transcription toproduce the 3′ untranslated region (3′UTR) of an mRNA molecule. The 3′untranslated region of an mRNA molecule may be generated by specificcleavage and 3′ polyadenylation, a.k.a. polyA tail. A 3′ UTR may beoperably linked to and located downstream of a transcribablepolynucleotide molecule and may include polynucleotides that provide apolyadenylation signal and other regulatory signals capable of affectingtranscription, mRNA processing, or gene expression. PolyA tails arethought to function in mRNA stability and in initiation of translation.Examples of 3′ transcription termination molecules in the art are thenopaline synthase 3′ region (see, Fraley, et al., Proc. Natl. Acad. Sci.USA, 80: 4803-4807 (1983)); wheat hsp17 3′ region; pea rubisco smallsubunit 3′ region; cotton E6 3′ region (U.S. Pat. No. 6,096,950); 3′regions disclosed in WO0011200A2; and the coixin 3′ UTR (U.S. Pat. No.6,635,806).

Constructs and vectors may also include a transit peptide codingsequence that expresses a linked peptide that is useful for targeting ofa protein product, particularly to a chloroplast, leucoplast, or otherplastid organelle; mitochondria; peroxisome; vacuole; or anextracellular location. For descriptions of the use of chloroplasttransit peptides, see U.S. Pat. Nos. 5,188,642 and 5,728,925. Manychloroplast-localized proteins are expressed from nuclear genes asprecursors and are targeted to the chloroplast by a chloroplast transitpeptide (CTP). Examples of such isolated chloroplast proteins include,but are not limited to, those associated with the small subunit (SSU) ofribulose-1,5,-bisphosphate carboxylase, ferredoxin, ferredoxinoxidoreductase, the light-harvesting complex protein I and protein II,thioredoxin F, enolpyruvyl shikimate phosphate synthase (EPSPS), andtransit peptides described in U.S. Pat. No. 7,193,133. It has beendemonstrated in vivo and in vitro that non-chloroplast proteins may betargeted to the chloroplast by use of protein fusions with aheterologous CTP and that the CTP is sufficient to target a protein tothe chloroplast. Incorporation of a suitable chloroplast transit peptidesuch as the Arabidopsis thaliana EPSPS CTP (CTP2) (See, Klee et al.,Mol. Gen. Genet., 210:437-442 (1987)) or the Petunia hybrida EPSPS CTP(CTP4) (See, della-Cioppa et al., Proc. Natl. Acad. Sci. USA,83:6873-6877 (1986)) has been show to target heterologous EPSPS proteinsequences to chloroplasts in transgenic plants (See, U.S. Pat. Nos.5,627,061; 5,633,435; and 5,312,910 and EP 0218571; EP 189707; EP508909; and EP 924299).

Transcribable Polynucleotide Molecules

As used herein, the term “transcribable polynucleotide molecule” refersto any DNA molecule capable of being transcribed into a RNA molecule,including, but not limited to, those having protein coding sequences andthose having sequences useful for gene suppression. A “transgene” refersto a transcribable polynucleotide molecule heterologous to a host celland/or a transcribable polynucleotide molecule artificially incorporatedinto a host cell's genome.

A promoter of the present invention may be operably linked to atranscribable polynucleotide molecule that is heterologous with respectto the promoter molecule. As used herein, the term “heterologous” refersto the combination of two or more polynucleotide molecules when such acombination would not normally be found in nature. For example, the twomolecules may be derived from different species and/or the two moleculesmay be derived from different genes, e.g. different genes from the samespecies or the same genes from different species. A promoter is thusheterologous with respect to an operably linked transcribablepolynucleotide molecule if such a combination is not normally found innature, i.e. that transcribable polynucleotide molecule is not naturallyoccurring operably linked in combination with that promoter molecule.

The transcribable polynucleotide molecule may generally be any DNAmolecule for which expression of an RNA transcript is desired. Suchexpression of an RNA transcript may result in translation of theresulting mRNA molecule and thus protein expression. Alternatively, atranscribable polynucleotide molecule may be designed to ultimatelycause decreased expression of a specific gene or protein. This may beaccomplished by using a transcribable polynucleotide molecule that isoriented in the antisense direction. One of ordinary skill in the art isfamiliar with using such antisense technology. Briefly, as the antisensetranscribable polynucleotide molecule is transcribed, the RNA producthybridizes to and sequesters a complementary RNA molecule inside thecell. This duplex RNA molecule cannot be translated into a protein bythe cell's translational machinery and is degraded in the cell. Any genemay be negatively regulated in this manner.

Thus, one embodiment of the invention is a regulatory element of thepresent invention, such as those provided as SEQ ID NO: 1-20, operablylinked to a transcribable polynucleotide molecule so as to modulatetranscription of the transcribable polynucleotide molecule at a desiredlevel or in a desired pattern upon introduction of said construct into aplant cell. In one embodiment, the transcribable polynucleotide moleculecomprises a protein-coding region of a gene, and the promoter affectsthe transcription of an RNA molecule that is translated and expressed asa protein product. In another embodiment, the transcribablepolynucleotide molecule comprises an antisense region of a gene, and thepromoter affects the transcription of an antisense RNA molecule or othersimilar inhibitory RNA molecule in order to inhibit expression of aspecific RNA molecule of interest in a target host cell.

Genes of Agronomic Interest

Transcribable polynucleotide molecules may be genes of agronomicinterest. As used herein, the term “gene of agronomic interest” refersto a transcribable polynucleotide molecule that when expressed in aparticular plant tissue, cell, or cell type provides a desirablecharacteristic associated with plant morphology, physiology, growth,development, yield, product, nutritional profile, disease or pestresistance, and/or environmental or chemical tolerance. Genes ofagronomic interest include, but are not limited to, those encoding ayield protein, a stress resistance protein, a developmental controlprotein, a tissue differentiation protein, a meristem protein, anenvironmentally responsive protein, a senescence protein, a hormoneresponsive protein, an abscission protein, a source protein, a sinkprotein, a flower control protein, a seed protein, an herbicideresistance protein, a disease resistance protein, a fatty acidbiosynthetic enzyme, a tocopherol biosynthetic enzyme, an amino acidbiosynthetic enzyme, a pesticidal protein, or any other agent such as anantisense or RNAi molecule targeting a particular gene for suppression.The product of a gene of agronomic interest may act within the plant inorder to cause an effect upon the plant physiology or metabolism or maybe act as a pesticidal agent in the diet of a pest that feeds on theplant.

In one embodiment of the invention, a promoter of the present inventionis incorporated into a construct such that the promoter is operablylinked to a transcribable polynucleotide molecule that is a gene ofagronomic interest. The expression of the gene of agronomic interest isdesirable in order to confer an agronomically beneficial trait. Abeneficial agronomic trait may be, for example, but not limited to,herbicide tolerance, insect control, modified yield, fungal diseaseresistance, virus resistance, nematode resistance, bacterial diseaseresistance, plant growth and development, starch production, modifiedoils production, high oil production, modified fatty acid content, highprotein production, fruit ripening, enhanced animal and human nutrition,biopolymers, environmental stress resistance, pharmaceutical peptidesand secretable peptides, improved processing traits, improveddigestibility, enzyme production, flavor, nitrogen fixation, hybrid seedproduction, fiber production, and biofuel production. Examples of genesof agronomic interest known in the art include those for herbicideresistance (U.S. Pat. Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114;6,107,549; 5,866,775; 5,804,425; 5,633,435; and 5,463,175), increasedyield (U.S. Pat. Nos. USRE38,446; 6,716,474; 6,663,906; 6,476,295;6,441,277; 6,423,828; 6,399,330; 6,372,211; 6,235,971; 6,222,098; and5,716,837), insect control (U.S. Pat. Nos. 6,809,078; 6,713,063;6,686,452; 6,657,046; 6,645,497; 6,642,030; 6,639,054; 6,620,988;6,593,293; 6,555,655; 6,538,109; 6,537,756; 6,521,442; 6,501,009;6,468,523; 6,326,351; 6,313,378; 6,284,949; 6,281,016; 6,248,536;6,242,241; 6,221,649; 6,177,615; 6,156,573; 6,153,814; 6,110,464;6,093,695; 6,063,756; 6,063,597; 6,023,013; 5,959,091; 5,942,664;5,942,658, 5,880,275; 5,763,245; and 5,763,241), fungal diseaseresistance (U.S. Pat. Nos. 6,653,280; 6,573,361; 6,316,407; 6,215,048;5,516,671; 5,773,696; 6,121,436; 6,316,407; and 6,506,962), virusresistance (U.S. Pat. Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864;5,850,023; and 5,304,730), nematode resistance (U.S. Pat. No.6,228,992), bacterial disease resistance (U.S. Pat. No. 5,516,671),plant growth and development (U.S. Pat. Nos. 6,723,897 and 6,518,488),starch production (U.S. Pat. Nos. 6,538,181; 6,538,179; 6,538,178;5,750,876; 6,476,295), modified oils production (U.S. Pat. Nos.6,444,876; 6,426,447; and 6,380,462), high oil production (U.S. Pat.Nos. 6,495,739; 5,608,149; 6,483,008; and 6,476,295), modified fattyacid content (U.S. Pat. Nos. 6,828,475; 6,822,141; 6,770,465; 6,706,950;6,660,849; 6,596,538; 6,589,767; 6,537,750; 6,489,461; and 6,459,018),high protein production (U.S. Pat. No. 6,380,466), fruit ripening (U.S.Pat. No. 5,512,466), enhanced animal and human nutrition (U.S. Pat. Nos.6,723,837; 6,653,530; 6,5412,59; 5,985,605; and 6,171,640), biopolymers(U.S. Pat. Nos. USRE37,543; 6,228,623; and 5,958,745, and 6,946,588),environmental stress resistance (U.S. Pat. No. 6,072,103),pharmaceutical peptides and secretable peptides (U.S. Pat. Nos.6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processingtraits (U.S. Pat. No. 6,476,295), improved digestibility (U.S. Pat. No.6,531,648) low raffinose (U.S. Pat. No. 6,166,292), industrial enzymeproduction (U.S. Pat. No. 5,543,576), improved flavor (U.S. Pat. No.6,011,199), nitrogen fixation (U.S. Pat. No. 5,229,114), hybrid seedproduction (U.S. Pat. No. 5,689,041), fiber production (U.S. Pat. Nos.6,576,818; 6,271,443; 5,981,834; and 5,869,720) and biofuel production(U.S. Pat. No. 5,998,700).

Alternatively, a gene of agronomic interest can affect the abovementioned plant characteristic or phenotype by encoding a RNA moleculethat causes the targeted modulation of gene expression of an endogenousgene, for example via antisense (see, e.g., U.S. Pat. No. 5,107,065);inhibitory RNA (“RNAi”, including modulation of gene expression viamiRNA-, siRNA-, trans-acting siRNA-, and phased sRNA-mediatedmechanisms, e.g., as described in published applications US2006/0200878, US 2008/0066206, and US2009/0070898; orcosuppression-mediated mechanisms. The RNA could also be a catalytic RNAmolecule (e.g., a ribozyme or a riboswitch; see e.g., US 2006/0200878)engineered to cleave a desired endogenous mRNA product. Thus, anytranscribable polynucleotide molecule that encodes a transcribed RNAmolecule that affects an agronomically important phenotype or morphologychange of interest may be useful for the practice of the presentinvention. Methods are known in the art for constructing and introducingconstructs into a cell in such a manner that the transcribablepolynucleotide molecule is transcribed into a molecule that is capableof causing gene suppression. For example, posttranscriptional genesuppression using a construct with an anti-sense oriented transcribablepolynucleotide molecule to regulate gene expression in plant cells isdisclosed in U.S. Pat. Nos. 5,107,065 and 5,759,829, andposttranscriptional gene suppression using a construct with asense-oriented transcribable polynucleotide molecule to regulate geneexpression in plants is disclosed in U.S. Pat. Nos. 5,283,184 and5,231,020. Expression of a transcribable polynucleotide in a plant cellcan also be used to suppress plant pests feeding on the plant cell, forexample, compositions isolated from coleopteran pests (U.S. PatentPublication No. US2007/0124836) and compositions isolated from nematodepests (U.S. Patent Publication No. US2007/0250947). Plant pests include,but are not limited to arthropod pests, nematode pests, and fungal ormicrobial pests. Exemplary transcribable polynucleotide molecules forincorporation into constructs of the present invention include, forexample, DNA molecules or genes from a species other than the targetspecies or genes that originate with or are present in the same species,but are incorporated into recipient cells by genetic engineering methodsrather than classical reproduction or breeding techniques. The type ofpolynucleotide molecule can include, but is not limited to, apolynucleotide molecule that is already present in the plant cell, apolynucleotide molecule from another plant, a polynucleotide moleculefrom a different organism, or a polynucleotide molecule generatedexternally, such as a polynucleotide molecule containing an antisensemessage of a gene, or a polynucleotide molecule encoding an artificial,synthetic, or otherwise modified version of a transgene.

Selectable Markers

As used herein the term “marker” refers to any transcribablepolynucleotide molecule whose expression, or lack thereof, can bescreened for or scored in some way. Marker genes for use in the practiceof the present invention include, but are not limited to transcribablepolynucleotide molecules encoding β-glucuronidase (GUS described in U.S.Pat. No. 5,599,670), green fluorescent protein and variants thereof (GFPdescribed in U.S. Pat. Nos. 5,491,084 and 6,146,826), proteins thatconfer antibiotic resistance, or proteins that confer herbicidetolerance. Useful antibiotic resistance markers, including thoseencoding proteins conferring resistance to kanamycin (nptII), hygromycinB (aph IV), streptomycin or spectinomycin (aad, spec/strep) andgentamycin (aac3 and aacC4) are known in the art. Herbicides for whichtransgenic plant tolerance has been demonstrated and the method of thepresent invention can be applied, include, but are not limited to:amino-methyl-phosphonic acid, glyphosate, glufosinate, sulfonylureas,imidazolinones, bromoxynil, dalapon, dicamba, cyclohexanedione,protoporphyrinogen oxidase inhibitors, and isoxaflutole herbicides.Transcribable polynucleotide molecules encoding proteins involved inherbicide tolerance are known in the art, and include, but are notlimited to, a transcribable polynucleotide molecule encoding5-enolpyruvylshikimate-3-phosphate synthase (EPSPS for glyphosatetolerance described in U.S. Pat. Nos. 5,627,061; 5,633,435; 6,040,497;and 5,094,945); a transcribable polynucleotide molecule encoding aglyphosate oxidoreductase and a glyphosate-N-acetyl transferase (GOXdescribed in U.S. Pat. No. 5,463,175; GAT described in U.S. Patentpublication No. 2003/0083480, and dicamba monooxygenase U.S. Patentpublication No. 2003/0135879); a transcribable polynucleotide moleculeencoding bromoxynil nitrilase (Bxn for Bromoxynil tolerance described inU.S. Pat. No. 4,810,648); a transcribable polynucleotide moleculeencoding phytoene desaturase (crtI) described in Misawa, et al., PlantJournal, 4:833-840 (1993) and Misawa, et al., Plant Journal, 6:481-489(1994) for norflurazon tolerance; a transcribable polynucleotidemolecule encoding acetohydroxyacid synthase (AHAS, aka ALS) described inSathasiivan, et al., Nucl. Acids Res., 18:2188-2193 (1990) for toleranceto sulfonylurea herbicides; and the bar gene described in DeBlock, etal., EMBO Journal, 6:2513-2519 (1987) for glufosinate and bialaphostolerance. The promoter molecules of the present invention can expresslinked transcribable polynucleotide molecules that encode forphosphinothricin acetyltransferase, glyphosate resistant EPSPS,aminoglycoside phosphotransferase, hydroxyphenyl pyruvate dehydrogenase,hygromycin phosphotransferase, neomycin phosphotransferase, dalapondehalogenase, bromoxynil resistant nitrilase, anthranilate synthase,aryloxyalkanoate dioxygenases, acetyl CoA carboxylase, glyphosateoxidoreductase, and glyphosate-N-acetyl transferase.

Included within the term “selectable markers” are also genes whichencode a secretable marker whose secretion can be detected as a means ofidentifying or selecting for transformed cells. Examples include markersthat encode a secretable antigen that can be identified by antibodyinteraction, or even secretable enzymes which can be detectedcatalytically. Selectable secreted marker proteins fall into a number ofclasses, including small, diffusible proteins which are detectable,(e.g., by ELISA), small active enzymes which are detectable inextracellular solution (e.g., alpha-amylase, beta-lactamase,phosphinothricin transferase), or proteins which are inserted or trappedin the cell wall (such as proteins which include a leader sequence suchas that found in the expression unit of extension or tobaccopathogenesis related proteins also known as tobacco PR-S). Otherpossible selectable marker genes will be apparent to those of skill inthe art and are encompassed by the present invention.

Cell Transformation

The invention is also directed to a method of producing transformedcells and plants which comprise a promoter operably linked to atranscribable polynucleotide molecule.

The term “transformation” refers to the introduction of nucleic acidinto a recipient host. As used herein, the term “host” refers tobacteria, fungi, or plant, including any cells, tissue, organs, orprogeny of the bacteria, fungi, or plant. Plant tissues and cells ofparticular interest include protoplasts, calli, roots, tubers, seeds,stems, leaves, seedlings, embryos, and pollen.

As used herein, the term “transformed” refers to a cell, tissue, organ,or organism into which a foreign polynucleotide molecule, such as aconstruct, has been introduced. The introduced polynucleotide moleculemay be integrated into the genomic DNA of the recipient cell, tissue,organ, or organism such that the introduced polynucleotide molecule isinherited by subsequent progeny. A “transgenic” or “transformed” cell ororganism also includes progeny of the cell or organism and progenyproduced from a breeding program employing such a transgenic organism asa parent in a cross and exhibiting an altered phenotype resulting fromthe presence of a foreign polynucleotide molecule. The term “transgenic”refers to a bacteria, fungi, or plant containing one or moreheterologous polynucleic acid molecules.

There are many methods for introducing polynucleic acid molecules intoplant cells. The method generally comprises the steps of selecting asuitable host cell, transforming the host cell with a recombinantvector, and obtaining the transformed host cell. Suitable methodsinclude bacterial infection (e.g. Agrobacterium), binary bacterialartificial chromosome vectors, direct delivery of DNA (e.g. viaPEG-mediated transformation, desiccation/inhibition-mediated DNA uptake,electroporation, agitation with silicon carbide fibers, and accelerationof DNA coated particles, etc. (reviewed in Potrykus, et al., Ann. Rev.Plant Physiol. Plant Mol. Biol., 42:205 (1991)).

Technology for introduction of a DNA molecule into cells is well knownto those of skill in the art. Methods and materials for transformingplant cells by introducing a plant DNA construct into a plant genome inthe practice of this invention can include any of the well-known anddemonstrated methods including, but not limited to:

-   -   (1) chemical methods (Graham and Van der Eb, Virology,        54:536-539 (1973) and Zatloukal, et al., Ann. N.Y. Acad. Sci.,        660:136-153 (1992));    -   (2) physical methods such as microinjection (Capecchi, Cell,        22:479-488 (1980)), electroporation (Wong and Neumann, Biochim.        Biophys. Res. Commun., 107:584-587 (1982); Fromm, et al, Proc.        Natl. Acad. Sci. USA, 82:5824-5828 (1985); U.S. Pat. No.        5,384,253) particle acceleration (Johnston and Tang, Methods        Cell Biol., 43(A):353-365 (1994); Fynan, et al., Proc. Natl.        Acad. Sci. USA, 90:11478-11482 (1993)): and microprojectile        bombardment (as illustrated in U.S. Pat. Nos. 5,015,580;        5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,403,865);    -   (3) viral vectors (Clapp, Clin. Perinatol., 20:155-168 (1993);        Lu, et al., J. Exp. Med., 178:2089-2096 (1993); Eglitis and        Anderson, Biotechniques, 6:608-614 (1988));    -   (4) receptor-mediated mechanisms (Curiel et al., Hum. Gen.        Ther., 3:147-154 (1992) and Wagner, et al., Proc. Natl. Acad.        Sci. USA, 89:6099-6103 (1992);    -   (5) bacterial mediated mechanisms such as Agrobacterium-mediated        transformation (as illustrated in U.S. Pat. Nos. 5,824,877;        5,591,616; 5,981,840; and 6,384,301);    -   (6) direct introduction into pollen by injecting a plant's        reproductive organs (Zhou, et al., Methods in Enzymology,        101:433, (1983); Hess, Intern Rev. Cytol., 107:367 (1987); Luo,        et al., Plant Mol. Biol. Reporter, 6:165 (1988); Pena, et al.,        Nature, 325:274 (1987));    -   (7) protoplast transformation (as illustrated in U.S. Pat. No.        5,508,184); and    -   (8) injection into immature embryos (Neuhaus, et al., Theor.        Appl. Genet., 75:30 (1987)).

Any of the above described methods may be utilized to transform a hostcell with one or more promoters and/or constructs of the present. Hostcells may be any cell or organism such as a plant cell, algae cell,algae, fungal cell, fungi, bacterial cell, or insect cell. Preferredhosts and transformed cells include cells from: plants, Aspergillus,yeasts, insects, bacteria and algae.

Methods for transforming dicotyledonous plants, primarily by use ofAgrobacterium tumefaciens and obtaining transgenic plants have beenpublished for cotton (U.S. Pat. Nos. 5,004,863; 5,159,135; and5,518,908); soybean (U.S. Pat. Nos. 5,569,834 and 5,416,011; see also,McCabe, et al., Biotechnology, 6:923 (1988) and Christou et al., PlantPhysiol. 87:671-674 (1988)); Brassica (U.S. Pat. No. 5,463,174); peanut(Cheng et al., Plant Cell Rep., 15:653-657 (1996) and McKently et al.,Plant Cell Rep., 14:699-703 (1995)); papaya; and pea (Grant et al.,Plant Cell Rep., 15:254-258 (1995)).

Transformations of monocotyledon plants using electroporation, particlebombardment, and Agrobacterium have also been reported. Transformationand plant regeneration have been achieved in asparagus (Bytebier, etal., Proc. Natl. Acad. Sci. U.S.A., 84:5354 (1987); barley (Wan andLemaux, Plant Physiol, 104:37 (1994)); maize (Rhodes, et al., Science240:204 (1988), Gordon-Kamm, et al., Plant Cell, 2:603-618 (1990),Fromm, et al., Bio/Technology, 8:833 (1990), Koziel et al.,Bio/Technology, 11:194 (1993), and Armstrong, et al., Crop Science,35:550-557 (1995)); oat (Somers, et al., Bio/Technology, 10:1589(1992)); orchard grass (Horn, et al., Plant Cell Rep. 7:469 (1988)); rye(De la Pena, et al., Nature, 325:274 (1987)); sugarcane (Bower andBirch, Plant Journal, 2:409 (1992)); tall fescue (Wang, et al.,Bio/Technology, 10:691 (1992)); and wheat (Vasil, et al.,Bio/Technology, 10:667 (1992) and U.S. Pat. No. 5,631,152).

The regeneration, development, and cultivation of plants fromtransformed plant protoplast or explants is well known in the art (see,for example, Weissbach and Weissbach, Methods for Plant MolecularBiology, (Eds.), Academic Press, Inc., San Diego, Calif. (1988) andHorsch et al., Science, 227:1229-1231 (1985)). Transformed cells aregenerally cultured in the presence of a selective media, which selectsfor the successfully transformed cells and induces the regeneration ofplant shoots and roots into intact plants (Fraley, et al., Proc. Natl.Acad. Sci. U.S.A., 80: 4803 (1983)). Transformed plants are typicallyobtained within two to four months.

The regenerated transgenic plants are self-pollinated to providehomozygous transgenic plants. Alternatively, pollen obtained from theregenerated transgenic plants may be crossed with non-transgenic plants,preferably inbred lines of agronomically important species. Descriptionsof breeding methods that are commonly used for different traits andcrops can be found in one of several reference books, see, for example,Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U. of CA,Davis, Calif., 50-98 (1960); Simmonds, Principles of crop improvement,Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen, Plant breedingperspectives, Wageningen (ed), Center for Agricultural Publishing andDocumentation (1979); Fehr, Soybeans: Improvement, Production and Uses,2nd Edition, Monograph., 16:249 (1987); Fehr, Principles of varietydevelopment, Theory and Technique, (Vol 1) and Crop Species Soybean (Vol2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987).Conversely, pollen from non-transgenic plants may be used to pollinatethe regenerated transgenic plants.

The transformed plants may be analyzed for the presence of the genes ofinterest and the expression level and/or profile conferred by theregulatory elements of the present invention. Those of skill in the artare aware of the numerous methods available for the analysis oftransformed plants. For example, methods for plant analysis include, butare not limited to Southern blots or northern blots, PCR-basedapproaches, biochemical analyses, phenotypic screening methods, fieldevaluations, and immunodiagnostic assays. The expression of atranscribable polynucleotide molecule can be measured using TaqMan®(Applied Biosystems, Foster City, Calif.) reagents and methods asdescribed by the manufacturer and PCR cycle times determined using theTaqMan® Testing Matrix. Alternatively, the Invader® (Third WaveTechnologies, Madison, Wis.) reagents and methods as described by themanufacturer can be used transgene expression.

The seeds of the plants of this invention can be harvested from fertiletransgenic plants and be used to grow progeny generations of transformedplants of this invention including hybrid plant lines comprising theconstruct of this invention and expressing a gene of agronomic interest.

The present invention also provides for parts of the plants of thepresent invention. Plant parts, without limitation, include leaves,stems, roots, tubers, seeds, endosperm, ovule, and pollen. The inventionalso includes and provides transformed plant cells which comprise anucleic acid molecule of the present invention.

The transgenic plant may pass along the transgenic polynucleotidemolecule to its progeny. Progeny includes any regenerable plant part orseed comprising the transgene derived from an ancestor plant. Thetransgenic plant is preferably homozygous for the transformedpolynucleotide molecule and transmits that sequence to all offspring asa result of sexual reproduction. Progeny may be grown from seedsproduced by the transgenic plant. These additional plants may then beself-pollinated to generate a true breeding line of plants. The progenyfrom these plants are evaluated, among other things, for geneexpression. The gene expression may be detected by several commonmethods such as western blotting, northern blotting,immuno-precipitation, and ELISA.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified. It should be appreciated bythose of skill in the art that the techniques disclosed in the followingexamples represent techniques discovered by the inventors to functionwell in the practice of the invention. However, those of skill in theart should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments that are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention, therefore all matter set forth or shown inthe accompanying drawings is to be interpreted as illustrative and notin a limiting sense.

EXAMPLES

Regulatory elements useful to drive expression of an operably linkedtranscribable polynucleotide in transgenic plants were isolated, and theexpression pattern of these regulatory elements operably linked to atranscribable polynucleotide molecule was analyzed in transgenic cornplants.

Example 1 Identification and Cloning of Regulatory Elements

Novel regulatory elements were identified and isolated from genomic DNAof the monocot species, Foxtail millet (Setaria italica (L.) Beauv). ESTsequence was used to design primers, which were then used withGenomeWalker™ (Clontech Laboratories, Inc, Mountain View, Calif.)libraries constructed following the manufacturer's protocol to clone the5′ region of the corresponding genomic DNA sequence. This cloned regioncontained the 5′ UTR sequence upstream of the protein-coding region foreach gene from S. italica. Using this sequence, regulatory elements werebioinformatically identified within the 5′ UTR for each gene.Bioinformatic analysis was used to identify the transcriptional startsite (TSS) and any bi-directionality, introns, or upstream codingsequence present in the sequence. Using the results of this analysis,regulatory elements were defined within the 5′ UTR sequence. Primerswere then designed to amplify the regulatory elements. The correspondingDNA molecule for each regulatory element was amplified using standardpolymerase chain reaction conditions with primers containing uniquerestriction enzyme sites and genomic DNA isolated from S. italica. Theresulting DNA fragments were ligated into a base plant expression vectorusing standard restriction enzyme digestion of compatible restrictionsites and DNA ligation methods. The resulting plant expression vectorscontained a right border region from Agrobacterium tumefaciens(B-AGRtu.right border), test regulatory element(s) operably linked to anintron derived from the HSP70 heat shock protein of Zea mays, operablylinked to a coding sequence for β-glucuronidase (GUS), operably linkedto the Nopaline synthase 3′ termination region from A. tumefaciens, anda left border region from A. tumefaciens (B-AGRtu.left border).

Sequences of the regulatory elements are provided herein as SEQ ID NO:1-20 and are listed in Table 1 below. Promoter sequences are providedherein as SEQ ID NO: 2, 5, 8, 10, 13, and 20. Leader sequences areprovided herein as SEQ ID NO: 3, 6, 11, and 16. Sequences providedherein as SEQ ID NO: 1, 4, 7, 9, 12, 14, 17, and 19 are an operablylinked promoter and leader sequence.

TABLE 1 Regulatory Elements. SEQ ID Annotation cDNA Annotation 1EXP-SETit.TIP Tonoplast Intrinsic Protein 2 P-SETit.Tip-1:1:1 TonoplastIntrinsic Protein 3 L-SETit.Tip-1:1:1 Tonoplast Intrinsic Protein 4EXP-SETit.Mtha Metallothionein-like protein 5 P-SETit.Mtha-1:1:1Metallothionein-like protein 6 L-SETit.Mth-1:1:1 Metallothionein-likeprotein 7 EXP-SETit.Mthb Metallothionein-like protein 8P-SETit.Mthb-1:1:2 Metallothionein-like protein 9 EXP-SETit.DRPaDehydration Related Protein a 10 P-SETit.DRPa-1:1:1 Dehydration RelatedProtein a 11 L-SETit.DRP-1:1:2 Dehydration Related Protein a/b 12EXP-SETit.DRPb Dehydration Related Protein b 13 P-SETit.DRPb-1:1:1Dehydration Related Protein b 14 EXP-SETit.Rcc3-1 Lipid Transfer Protein15 P-SETit.Rcc3-1:1:1 Lipid Transfer Protein 16 L-SETit.Rcc3-1:1:2 LipidTransfer Protein 17 EXP-SETit.Rcc3-10 Lipid Transfer Protein 18P-SETit.Rcc3-1:1:10 Lipid Transfer Protein 19 EXP-SETit.Rcc3-11 LipidTransfer Protein 20 P-SETit.Rcc3-1:1:11 Lipid Transfer Protein

The expression element, EXP-SETit.TIP (SEQ ID NO: 1) is comprised of theP-SETit.Tip-1:1:1 promoter (SEQ ID NO: 2) and the L-SETit.Tip-1:1:1leader (SEQ ID NO: 3).

For the regulatory elements from the Lipid Transfer Protein gene, threepromoter variants were designed (FIG. 1). P-SETit.Rcc3-1:1:1 a 2062nucleotide version (SEQ ID NO:15); P-SETit.Rcc3-1:1:10 a 1563 nucleotideversion (SEQ ID NO:18); and P-SETit.Rcc3-1:1:11 a 915 nucleotide version(SEQ ID NO:20). The expression element, EXP-SETit.Rcc3-1 (SEQ ID NO: 14)is comprised of the P-SETit.Rcc3-1:1:1 promoter (SEQ ID NO: 15) and theP-SETit.Rcc3-1:1:2 leader (SEQ ID NO: 16). The expression element,EXP-SETit.Rcc3-10 (SEQ ID NO: 17) is comprised of theP-SETit.Rcc3-1:1:10 promoter (SEQ ID NO: 18) and the L-SETit.Rcc3-1:1:2leader (SEQ ID NO: 16). The expression element, EXP-SETit.Rcc3-11 (SEQID NO: 19) is comprised of the P-SETit.Rcc3-1:1:11 promoter (SEQ ID NO:20) and the P-SETit.Rcc3-1:1:2 leader (SEQ ID NO: 16).

For the regulatory elements from the Metallothionein-like protein gene,two promoter variants were designed (FIG. 2). P-SETit.Mtha-1:1:1 is ashorter 483 nucleotide version; P-SETit.Mthb-1:1:2 is a longer 1516nucleotide version of this. The expression element, EXP-SETit.Mtha (SEQID NO: 4) is comprised of the P-SETit.Mtha-1:1:1 promoter (SEQ ID NO: 5)and the L-SETit.Mth-1:1:1 leader (SEQ ID NO: 6). The expression element,EXP-SETit.Mthb (SEQ ID NO: 7) is comprised of the P-SETit.Mthb-1:1:2promoter (SEQ ID NO: 8) and the L-SETit.Mth-1:1:1 leader (SEQ ID NO: 6).

For the regulatory elements from the Dehydration Related Protein gene,the regulatory elements from two allelic variants were isolated (FIGS.3A and 3B). The two allelic variants have identical leader sequences,but the promoter sequences are variants with several base changes andinsertion/deletions when aligned. The expression element, EXP-SETit.DRPa(SEQ ID NO: 9) is comprised of the P-SETit.DRP-1:1:1 promoter (SEQ IDNO: 10) and the L-SETit.DRP-1:1:2 leader (SEQ ID NO: 11). The expressionelement, EXP-SETit.DRPb (SEQ ID NO: 12) is comprised of theP-SETit.DRPb-1:1:1 promoter (SEQ ID NO: 13) and the L-SETit.DRP-1:1:2leader (SEQ ID NO: 11).

Example 2 Analysis of Regulatory Elements Driving Gus in Transgenic Corn

Corn plants were transformed with plant expression vectors containingthe test regulatory elements driving expression of the β-glucuronidase(GUS) transgene, and the resulting plants were analyzed for GUS proteinexpression.

Corn plants were transformed with plant GUS expression constructs,pMON101552 (EXP-SETit.TIP, SEQ ID NO: 1), pMON99662 (EXP-SETit.Mtha, SEQID NO: 4), and pMON99663 (EXP-SETit.DRPa, SEQ ID NO: 9). Plants weretransformed using particle bombardment methods known to those skilled inthe art and corn H99 immature zygotic embryos to produce transgenicmaize plants. Briefly, ears of maize H99 plants were collected 10-13days after pollination from greenhouse-grown plants and sterilized.Immature zygotic embryos of 1.2-1.5 mm were excised from the ear andincubated at 28° Celsius in the dark for 3-5 days before use as targettissue for bombardment. The plant transformation vector containing theselectable marker for kanamycin resistance (NPTII gene) and the GUSexpression cassette was digested with restriction endonucleases. Asingle DNA fragment containing both the selectable marker and GUSexpression cassette was gel purified and used to coat 0.6 micron goldparticles (Catalog #165-2262 Bio-Rad, Hercules, Calif.) for bombardment.Macro-carriers were loaded with the DNA-coated gold particles (Catalog#165-2335 Bio-Rad, Hercules Calif.). The embryos were transferred ontoosmotic medium, scutellum side up. A PDS1000/He biolistic gun was usedfor transformation (Catalog #165-2257 Bio-Rad, Hercules Calif.).Bombarded immature embryos were cultured and transgenic calli wereselected and transferred to tissue regeneration medium. Transgenic cornplants were regenerated from the transgenic calli and transferred to thegreenhouse.

Histochemical GUS analysis was used for qualitative expression analysisof transformed plants. Whole tissue sections were incubated with GUSstaining solution X-Gluc (5-bromo-4-chloro-3-indolyl-b-glucuronide) (1milligram/milliliter) for an appropriate length of time, rinsed, andvisually inspected for blue coloration. GUS activity was qualitativelydetermined by direct visual inspection or inspection under a microscopeusing selected plant organs and tissues. The R₀ plants were inspectedfor expression in the roots and leaves. The regulatory elementsEXP-SETit.TIP (SEQ ID NO: 1), EXP-SETit.Mtha (SEQ ID NO: 4), andEXP-SETit.DRPa (SEQ ID NO: 9) demonstrated GUS expression in both rootsand leaves in the R₀ transformants.

Plants transformed with GUS expression driven by either EXP-SETit.TIP(SEQ ID NO: 1) or EXP-SETit.Mtha (SEQ ID NO: 4) were crossed withnon-transformed H99 plants to produce an F₁ population of transformants.GUS expression levels were measured in selected tissues over the courseof development. The F₁ tissues used for this study included: imbibedseed embryo, imbibed seed endosperm, root (3 days after germination),coleoptiles (3 days after germination), V3 main and crown root, V3 leaf,V7 seminal and crown root, V7 mature leaf, VT (at tasseling, prior toreproduction) seminal root, VT internode, VT cob, VT anther, VT pollen,VT silk, kernel 7 days after pollination, embryo on 21 days afterpollination, endosperm 21 days after pollination, embryo 35 days afterpollination, endosperm 35 days after pollination. F₁ GUS expression wasseen in all tissues studied for both for events transformedEXP-SETit.TIP (SEQ ID NO: 1) and EXP-SETit.Mtha (SEQ ID NO: 4).

Example 3 Analysis of Regulatory Elements Driving TIC809 in TransgenicCorn

Corn plants were transformed with plant expression vectors containingthe test regulatory elements driving expression of the TIC809 transgene,and the resulting plants were analyzed for TIC809 protein expression.

The regulatory elements were operably linked to the insect toxintransgene, TIC809 (PCTUS2006/033867) in plant transformation vectors.The transgene cassette was comprised of regulatory element(s) operablylinked to an intron derived from the HSP70 heat shock protein of Zeamays, operably linked to the TIC809 transgene, operably linked to a 3′UTR derived from the Triticum aestivum L. HSP17 gene. The planttransformation vectors also contained a glyphosate tolerance selectablemarker for selection of transformed plant cells.

Corn tissue from variety LH244 was transformed using A. tumefaciensmediated transformation with plasmids, pMON70539 (EXP-SETit.TIP, SEQ IDNO: 1), pMON70540 (EXP-SETit.Mtha, SEQ ID NO: 4), and pMON70538(EXP-SETit.DRPa, SEQ ID NO: 9) using methods known in the art. R₀ plantswere regenerated from the transformed corn tissue and tested to confirmthe presence and intactness of the TIC809 transgene. Leaves and rootsfrom these plants were analyzed at the V4 or V6 stage using a TIC809Enzyme-Linked ImmunoSorbent Assay (ELISA) to determine the levels ofTIC809 protein accumulation. Protein values were determined using aTIC809 reference sample and expressed in units of parts per million(ppm). ELISA data is presented in Table 2 below where “N” indicates thenumber of plants tested.

TABLE 2 TIC809 R₀ ELISA Data. Root Leaf Element Name SEQ ID NO Vector(ppm) (ppm) N EXP-SETit.TIP 1 pMON70539 3 6 26 EXP-SETit.Mtha 4pMON70540 3 8 20 EXP-SETit.DRPa 9 pMON70538 1 2 24

TIC809 expression, driven by the expression elements, EXP-SETit.TIP (SEQID NO: 1), EXP-SETit.Mtha (SEQ ID NO: 4), and EXP-SETit.DRPa (SEQ ID NO:9) was seen in both roots and leaves, thus demonstrating the ability ofthe regulatory elements EXP-SETit.TIP, EXP-SETit.Mtha, andEXP-SETit.DRPa to modulate transcription of an operably linked transgeneof agronomic interest in plants. The average TIC809 expression driven byEXP-SETit.TIP (SEQ ID NO: 1), EXP-SETit.Mtha (SEQ ID NO: 4), andEXP-SETit.DRPa (SEQ ID NO: 9) was at least 2 fold or higher in the leafrelative to root for all three constructs.

Transformed R0 plants containing the vectors pMON70539 (EXP-SETit.TIP,SEQ ID NO: 1), pMON70540 (EXP-SETit.Mtha, SEQ ID NO: 4), and pMON70538(EXP-SETit.DRPa, SEQ ID NO: 9) were crossed with variety LH59, usingLH59 as the female plant and transformed LH244 as the male, to producetransformed F₁ populations. For plants containing EXP-SETit.TIP (SEQ IDNO: 1) and EXP-SETit.Mtha (SEQ ID NO: 4), TIC809 protein levels werethen measured using ELISA in F₁ leaves at V3, V7, and VT stages; inroots at V3 and V7 stages; in reproductive tissues around VT stage(anther, pollen and silk); and in the developing seed or kernel. TheELISA results are expressed in parts per million (ppm) with standarderror measurements indicated as “SE” and presented in Table 3 below. Forplants containing EXP-SETit.DRPa (SEQ ID NO: 9), TIC809 protein levelswere measured in roots using ELISA with tissue taken at V9 stage. TheELISA results are expressed in parts per million (ppm) and presented inTable 4 below.

TABLE 3 TIC809 F₁ ELISA Data for EXP-SETit.TIP and EXP-SETit.Mtha. EXP-EXP- SETit.TIP SETiT.Mtha Tissue ppm SE ppm SE Leaf V3 1.138 0.32 1.3120.46 Root V3 1.254 0.41 1.242 0.39 Stalk V3 0.694 0.14 0.407 0.19 LeafV7 0.879 0.31 1.295 0.31 Root V7 0.755 0.35 1.185 0.46 Leaf VT 0.3080.17 0.457 0.35 Anther 0.323 0.06 0.364 0.1 Silk 0.249 0.1 0.222 0.05Pollen 0.315 0.16 0.231 0.01 Seed 0.138 0.02 0 0

Average TIC809 protein expression levels driven by the regulatoryelements, EXP-SETit.TIP (SEQ ID NO: 1) and EXP-SETit.Mtha (SEQ ID NO: 4)in the F1 populations were consistent with the expression levelsobserved in the R0 transformants, confirming the ability of the TIP andMtha regulatory elements to drive expression of an operably linkedtransgene. EXP-SETit.TIP (SEQ ID NO: 1) appears to have its highestlevel of expression at V3 in the roots and leaves, with expressiondeclining by V7 stage and low in the reproductive tissues and developingseed. EXP-SETit.Mtha (SEQ ID NO: 4) shows consistent expression ofTIC809 in the roots and leaves between V3 and V7 stage, with a declinein expression in the VT stage leaf, lower expression in the reproductivetissue, and no expression in the developing seed.

TABLE 4 TIC809 F1 ELISA Data for EXP-SETit.DRPa. F₁ Cross Tissue ppmLH59/ZM_S198227 0.95 LH59/ZM_S198230 2.69 LH59/ZM_S198235 0.5LH59/ZM_S199429 1.26 LH59/ZM_S201032 1.09 LH59/ZM_S201049 0.64 AverageTIC809 1.19 Expression Standard Error 0.79

Average TIC809 protein expression levels driven by the regulatoryelement, EXP-SETit.DRPa (SEQ ID NO: 9) in the F1 population roots wereconsistent with the expression levels observed in the R₀ transformants,confirming the ability of the DRPa regulatory elements to provide rootexpression of an operably linked transgene.

R₀ populations of plants transformed with pMON120408 (EXP-SETit.Rcc3-1,SEQ ID NO: 14), pMON120407 (EXP-SETit.Rcc3-10, SEQ ID NO: 17), andpMON120410 (EXP-SETit.Rcc3-11, SEQ ID NO: 19) were produced as describedabove. TIC809 protein levels in these plants were measured in leaves androots using ELISA with tissue taken at the V4 or V6 stage. The ELISAresults are expressed in parts per million (ppm) and are presented inTable 5 below.

TABLE 5 TIC809 R₀ ELISA Data for EXP-SETit.Rcc3-1, EXP-SETit.Rcc3-10,and EXP-SETit.Rcc3-11. Root Leaf Element Name Event (ppm) (ppm)EXP-SETit.Rcc3-1 (SEQ ID NO: 14) 1 0.292 0 EXP-SETit.Rcc3-1 (SEQ ID NO:14) 2 2.79 0 EXP-SETit.Rcc3-1 (SEQ ID NO: 14) 3 1.171 0 EXP-SETit.Rcc3-1(SEQ ID NO: 14) Average 1.42 0 EXP-SETit.Rcc3-10 (SEQ ID NO: 17) 1 2.1290 EXP-SETit.Rcc3-10 (SEQ ID NO: 17) 2 1.793 0 EXP-SETit.Rcc3-10 (SEQ IDNO: 17) 3 0.425 0 EXP-SETit.Rcc3-10 (SEQ ID NO: 17) 4 0.677 0EXP-SETit.Rcc3-10 (SEQ ID NO: 17) 5 0.877 0 EXP-SETit.Rcc3-10 (SEQ IDNO: 17) 6 1.847 0 EXP-SETit.Rcc3-10 (SEQ ID NO: 17) 7 1.479 0EXP-SETit.Rcc3-10 (SEQ ID NO: 17) 8 0.359 0 EXP-SETit.Rcc3-10 (SEQ IDNO: 17) 9 1.84 0 EXP-SETit.Rcc3-10 (SEQ ID NO: 17) 10 3.309 0EXP-SETit.Rcc3-10 (SEQ ID NO: 17) 11 1.589 0 EXP-SETit.Rcc3-10 (SEQ IDNO: 17) 12 3.652 0.294 EXP-SETit.Rcc3-10 (SEQ ID NO: 17) 13 1.019 0EXP-SETit.Rcc3-10 (SEQ ID NO: 17) 14 2.715 0.56 EXP-SETit.Rcc3-10 (SEQID NO: 17) 15 0.981 0 EXP-SETit.Rcc3-10 (SEQ ID NO: 17) 16 3.147 0.317EXP-SETit.Rcc3-10 (SEQ ID NO: 17) Average 1.74 0.07 EXP-SETit.Rcc3-11(SEQ ID NO: 19) 1 2.356 0 EXP-SETit.Rcc3-11 (SEQ ID NO: 19) 2 2.343 0EXP-SETit.Rcc3-11 (SEQ ID NO: 19) 3 1.465 0 EXP-SETit.Rcc3-11 (SEQ IDNO: 19) 4 2.271 0 EXP-SETit.Rcc3-11 (SEQ ID NO: 19) 5 0.247 0EXP-SETit.Rcc3-11 (SEQ ID NO: 19) 6 2.949 0 EXP-SETit.Rcc3-11 (SEQ IDNO: 19) 7 3.36 0 EXP-SETit.Rcc3-11 (SEQ ID NO: 19) 8 4.771 0EXP-SETit.Rcc3-11 (SEQ ID NO: 19) 9 1.757 0 EXP-SETit.Rcc3-11 (SEQ IDNO: 19) 10 2.255 0 EXP-SETit.Rcc3-11 (SEQ ID NO: 19) 11 3.314 0EXP-SETit.Rcc3-11 (SEQ ID NO: 19) 12 4.958 0 EXP-SETit.Rcc3-11 (SEQ IDNO: 19) 13 0.232 0 EXP-SETit.Rcc3-11 (SEQ ID NO: 19) 14 1.503 0EXP-SETit.Rcc3-11 (SEQ ID NO: 19) 15 1.425 0 EXP-SETit.Rcc3-11 (SEQ IDNO: 19) 16 3.6 0 EXP-SETit.Rcc3-11 (SEQ ID NO: 19) 17 3.533 0EXP-SETit.Rcc3-11 (SEQ ID NO: 19) 18 2.158 0 EXP-SETit.Rcc3-11 (SEQ IDNO: 19) Average 2.47 0

Expression of TIC809 when driven by the expression elements variantsEXP-SETit.Rcc3-1(SEQ ID NO: 14), EXP-SETit.Rcc3-10 (SEQ ID NO: 17), andEXP-SETit.Rcc3-11 (SEQ ID NO: 19) was strong in roots and weak or absentin leaves. Low or no leaf expression was observed for the threeSETit.Rcc3 variants with the exception of three transformed events inwhich the expression element variant, EXP-SETit.Rcc3-10 (SEQ ID NO: 17)showed a low level of TIC809 expression in leaves. The average rootexpression of the TIC809 protein was higher using the expression elementvariant, EXP-SETit.Rcc3-11 (SEQ ID NO: 19) than the EXP-SETit.Rcc3-1(SEQID NO: 14) and EXP-SETit.Rcc3-10 (SEQ ID NO: 17) variants, but all threevariants provided root expression of the operably linked transgene.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the claims. All publications and published patentdocuments cited herein are hereby incorporated by reference to the sameextent as if each individual publication or patent application isspecifically and individually indicated to be incorporated by reference.

We claim:
 1. A DNA molecule comprising a regulatory element having a DNAsequence selected from the group consisting of: (a) the sequence of SEQID NO:5, and (b) a fragment of at least 95 contiguous nucleotides of SEQID NO:5 with promoter activity, wherein said regulatory element isoperably linked to a heterologous transcribable polynucleotide molecule.2. The DNA molecule of claim 1, wherein the transcribable polynucleotideis a sequence of agronomic interest.
 3. The DNA molecule of claim 1,wherein the transcribable polynucleotide is a sequence capable ofproviding herbicide resistance in plants.
 4. The DNA molecule of claim1, wherein the transcribable polynucleotide is a sequence capable ofproviding plant pest control in plants.
 5. A transgenic plant cellstably transformed with the DNA molecule of claim
 1. 6. The transgenicplant cell of claim 5, wherein said transgenic plant cell is amonocotyledonous plant cell.
 7. A transgenic plant stably transformedwith the DNA molecule of claim
 1. 8. A plant part of the transgenicplant of claim 7, wherein the plant part comprises the DNA molecule. 9.A seed of the transgenic plant of claim 7, wherein the seed comprisesthe DNA molecule.
 10. The DNA molecule of claim 1, wherein the DNAmolecule comprises SEQ ID NO:5.
 11. The DNA molecule of claim 1, whereinthe fragment comprises at least 250 contiguous nucleotides of SEQ IDNO:5.
 12. The DNA molecule of claim 1, wherein the fragment comprises atleast 500 contiguous nucleotides of SEQ ID NO:5.
 13. The DNA molecule ofclaim 1, wherein the fragment comprises at least 750 contiguousnucleotides of SEQ ID NO:5.
 14. The transgenic plant of claim 7, whereinthe DNA molecule comprises SEQ ID NO:5.
 15. The transgenic plant ofclaim 7, wherein the fragment comprises at least 250 contiguousnucleotides of SEQ ID NO:5.
 16. The transgenic plant of claim 7, whereinthe fragment comprises at least 500 contiguous nucleotides of SEQ IDNO:5.
 17. The transgenic plant of claim 7, wherein the fragmentcomprises at least 750 contiguous nucleotides of SEQ ID NO:5.
 18. Theseed of claim 9, wherein the DNA molecule comprises SEQ ID NO:5.
 19. Theseed of claim 9, wherein the fragment comprises at least 250 contiguousnucleotides of SEQ ID NO:5.
 20. The seed of claim 9, wherein thefragment comprises at least 500 contiguous nucleotides of SEQ ID NO:5.21. The seed of claim 9, wherein the fragment comprises at least 750contiguous nucleotides of SEQ ID NO:5.